Kallikrein 5 1536 HTS Dose Response Confirmation
Human kallikrein 5 (hK5) is a member of the human tissue kallikrein family, which contains 15 kallikrein-like serine proteases (1). It is synthesized as a 293 amino acid zymogen, and loses a 29 amino acid signal peptide upon secretion, followed by cleavage at the Arg66-Ile67 bond, which releases a 37 amino acid activation peptide, resulting in a 237 amino acid mature enzyme (2). hK5 generally more ..
BioActive Compounds: 109
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01
Human kallikrein 5 (hK5) is a member of the human tissue kallikrein family, which contains 15 kallikrein-like serine proteases (1). It is synthesized as a 293 amino acid zymogen, and loses a 29 amino acid signal peptide upon secretion, followed by cleavage at the Arg66-Ile67 bond, which releases a 37 amino acid activation peptide, resulting in a 237 amino acid mature enzyme (2). hK5 generally exhibits a trypsin-like specificity for P1-Arg over P1-Lys residues, and has been observed to digest different extracellular matrix and plasma proteins (3, 4). Protein inhibitors include alpha2-antiplasmin, antithrombin III and alpha2-macroglobulin (4). In addition, Zn2+ and other divalent cations strongly inhibit hK5 (5).
hK5 is highly expressed in skin tissue, specifically the outermost layer of the skin, the stratum corneum, but is found in other tissues, including brain, placenta, and kidney (2). Within the stratum corneum, hK5 is found in the upper spinous and granular layers, where keratinocytes undergo turnover and differentiation into flattened structures called corneocytes that make up the stratum corneum (6, 7). It has also been characterized as a marker for ovarian and breast cancer, having previously been shown to be differentially regulated in several different hormone-dependent malignancies (8, 9).
Recently, hK5 was found to be involved in the common, chronic skin condition rosacea (10). This condition involves dilation of blood vessels near the surface of the skin, resulting in erythema accompanied by visible blood vessels along the middle of the face. Abnormal cutaneous vasculature is often cited as an underlying cause, but immune responses associated with microbes, and the resultant acne, have also been suggested as causes. To this end, Yamasaki et al. report that high levels of the antimicrobial peptide cathelicidin were found in rosacea lesions, suggesting that abnormal innate immune responses are to blame (10). High levels of hK5 expression were also found, and its expression was colocalized with cathilicidin, while lower levels were observed on normal skin. In situ zymography showed that protease activity overall was elevated in rosacea, while absent in normal skin, and the serine protease inhibitors aprotinin and AEBSF suppressed the protease activity in rosacea lesions. Thus, isolation of inhibitors against hK5, as well as other proteases from the skin, could allow for development of a therapeutic topical agent that would alleviate rosacea.
HTS was performed using 214,203 compounds of the MLSCN library individually plated into 10ul 1536 compound plates at a concentration of 2.5 mM each, which were diluted 500-fold into 5 ul 1536 well assay plates (final concentration 5 uM each compound). The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of Boc-Vsl-Pro-Arg-AMC, as first described by Michael et al. (4).
1.E. P. Diamandis et al., Clin Chem 46, 1855 (Nov, 2000).
2.M. Brattsand, T. Egelrud, J Biol Chem 274, 30033 (Oct 15, 1999).
3.M. Debela et al., J Biol Chem 281, 25678 (Sep 1, 2006).
4.I. P. Michael et al., J Biol Chem 280, 14628 (Apr 15, 2005).
5.I. P. Michael et al., J Biol Chem 281, 12743 (May 5, 2006).
6.A. Ishida-Yamamoto et al., J Invest Dermatol 124, 360 (Feb, 2005).
7.B. Sondell, L. E. Thornell, T. Egelrud, J Invest Dermatol 104, 819 (May, 1995).
8.E. P. Diamandis et al., Tumour Biol 24, 299 (Nov-Dec, 2003).
9.G. M. Yousef et al., Cancer Res 63, 3958 (Jul 15, 2003).
10.K. Yamasaki et al., Nat Med 13, 975 (Aug, 2007).
Human kallikrein 5 (hK5) was purchased from R&D Systems (Cat #1108-SE). Substrate Boc-Val-Pro-Arg-AMC was from Bachem (Cat #I-1120.0050). Assay buffer consisted of 50 mM Tris, pH 8.0, 0.05% Tween 20. 1536-well black plates were from Corning (Item #3728).
hK5 (0.60 ug/mL) was incubated with Boc-Phe-Ser-Arg-AMC substrate (15 uM) in 5 uL of assay buffer (see above) for 2 hr at room temperature. HTS was performed using 5 uM compound.
1.Serial dilute single compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2.Fill low-volume plate with 4 uL water using Multidrop-micro
3.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
4.Add 200 nL of compound (in DMSO from step 1) using 2 transfers of 100 nL with the Evolution 384 pintool (washed with isopropanol after each transfer)
5.Add 1 uL of Boc-Val-Pro-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (1.35 ug/mL in assay buffer) using Multidrop-384
7.Incubate for 3 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader
IC50 plates contained compounds in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 1.5 nM. Percent activity was calculated for each dilution of each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:
% Activity = 100*((signal-blank mean)/(control mean-blank mean))
Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively).
The activity score reported here is based on follow-up IC50 testing on compounds that showed >40% inhibition in the primary HTS:
IC50 scores were calculated as follows:
(1) Score = 5.75 x (pIC50), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >50 uM, the score was calculated from percent activity at maximum concentration tested in assay (50 uM):
Score = [5.75 x (0-3)] + [(100-percent activity at max concentration)/1.75]
Compounds that gave percent inhibition >40% in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM.
Activity outcome is reported as follows:
(1) IC50 <50 uM = active
(2) IC50 >50 uM, percent inhibition 30-50% at 50 uM = inconclusive
(3) IC50 >50 uM, percent inhibition <30% at 50 uM = inactive
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Paul Riley and Sahil Batta, and data were submitted by Andrew Napper and Paul Riley, all of the University of Pennsylvania.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)